The serotonin (5HT) transporter (SERT) is a clinically important target for antidepressants and psychostimulants, including cocaine and 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), but much remains to be defined regarding SERT substrate binding and translocation. The recent elucidation of a crystal structure of a bacterial homology of SERT, the leucine transporter LeuTAa, provides an important tool for the elucidation of sites defining SERT substrate selectivity (Yamashita et al., 2005). Homology modeling of the human SERT (hSERT) protein onto the LeuTAa backbone predicts that transmembrane domains (TMs) 1, 3, 6, and 8 likely comprise the 5HT binding pocket. Of these, TM 6 is predicted to possess important determinants of 5HT interaction but has yet to be explored biochemically and pharmacologically. To this end, both model-guided and model-independent approaches will be employed to define substrate accessibility and critical binding residues within hSERT TM6. The aqueous accessibility of TM6 residues will be investigated using the substituted cysteine accessibility method (SCAM), in which individual cysteine substitution mutants are probed with methanethiosulfonate (MTS) reagents and then assayed for uptake activity. Controls will include alanine mutagenesis of exposed residues and MTSEA-biotinylation. Residues that are shown to be sensitive to MTS inactivation will be further examined for potential interactions with substrates (5HT and MDMA), ions (sodium and chloride), and inhibitors (cocaine, citalopram and paroxetine). MTS protection assays will be utilized to identify TM6 residues that may participate in ligand binding and selectivity. Comparisons of 5HT to MDMA will be made to assess whether psychostimulantspecific conformations of TM6 exist to sustain 5HT efflux. Residues implicated by these MTS protection assays will be subjected to additional mutagenesis to further investigate the contributions of individual side chain character to substrate interaction. Finally, a library of tryptamine derivatives and other substrate analogs will be screened against selected TM6 mutants in order to develop a better understanding of 5HTs orientation within the binding pocket. This examination of the serotonin transporter will contribute to a better definition of substrate and antagonist binding determinants. An understanding of how drugs like cocaine and MDMA act at the transporter will aid endeavors to develop strategies targeted at reducing the effects of these drugs of abuse. [unreadable] [unreadable] [unreadable]